In this application, we propose studies that will enable us to retrieve cancer cells that are hypoxic and/or cycling from tumor xenografts using our newly developed pair of fluorescent protein reporter system that we engineered and termed HypoxCR (Hypoxia - cell Cycle Reporters). With HypoxCR, we will study a panel of human cancer cell lines in vitro and in vivo to characterize their genomic and metabolic reprogramming upon exposure to hypoxia in vitro or in tumor xenografts. The delineation of the genomic and metabolic programs of the subpopulations of cancer cells in a tumor will instruct us about the commensal relations amongst cancer cells in a tumor tissue and lead us to novel therapeutic targets in addition to candidates that we identified from Myc and/or HIF targets, such as lactate dehydrogenase A (LDHA) and glutatminase (GLS). Our long term goal is to identify molecular targets from the reprogramming of cancer cells in the tumor tissue and find potential drug-like inhibitors of these targets as experimental molecular probes and potential therapeutic agents. To reach our goals, we set the following Aims: Aim 1. To characterize cancer cells in the tumor microenvironment with a novel dual fluorescent protein reporter system for hypoxia and cell cycling (HypoxCR). Using this double reporter system, we propose to retrieve cancer cells from different subpopulations of the tumor xenograft to characterize their genomic changes and phenotypes and compare these changes with those arising in vitro under hypoxia. Aim 2. To delineate the genomic reprogramming of cancer cells by MYC and HIF-1 in vitro and in vivo. Using in vitro models of tumor hypoxia and cells retrieved from xenografts marked by HypoxCR, we will use high throughput microarray-based methods to delineate the genomic reprogramming of cells by MYC and HIF-1 in the tumor microenvironment. Aim3. To determine the role of key nodal points in metabolic reprogramming of cancer cells in hypoxia and in the tumor microenvironment. We propose to study miR-23a/b (which is suppressed by Myc and regulates glutaminase protein levels) and MCT1 (monocarboxylate/lactate transporter) in the reprogramming of cancer cells in the tumor microenvironment. Aim 4. To determine the effects of inhibitors of key metabolic nodes in cancer cells in vitro and in vivo. We propose to use fluorescent HypoxCR reporters to determine the sensitivity of subpopulations of tumor cells to therapy in vivo and establish complementation between drug-like molecules (eg., LDHA and GLS inhibitors) that target different tumor cell subpopulations.